Salt of (2s, 3s) -3- [ [ (1s) -1- isobutoxymethyl-3-methylbutyl] carbamyl] oxirane-2-carboxylic acid

ABSTRACT

The invention relates to purification of (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid [hereinafter, referred to as the carboxylic acid] using the salt of the carboxylic acid with an organic amine selected from the group consisting of piperazine, adamantane amines and others. The invention also relates to providing crystalline sodium salt of the carboxylic acid. The sodium salt is usable as a material for preparing medicaments and improved in storage stability, and has the following characteristics:
         DSC: exothermic peak observed at a temperature in the range of 170 to 175° C. with weight decrease; and   characteristic absorption bands of infrared absorption spectrum (KBr tablet): 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365, 1310, 1260, 1110, 890 cm ×1 .

FIELD OF THE INVENTION

The present invention relates to a salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylicacid.

BACKGROUND OF THE INVENTION

The below-illustrated sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylate(hereinafter referred to as Compound A) shows a cathepsin-inhibitoryaction and is useful as a remedy for treating rheumatoid arthritis andosteoporosis (Patent reference 1: WO 99/11640 pamphlet):

Patent reference 1 describes a process for producing Compound Aaccording to the following reaction scheme (see Example 48 of Patentreference 1):

The starting ester material of the above-mentioned reaction scheme isgenerally purified by column chromatography to obtain a purifiedCompound A.

DISCLOSURE OF THE INVENTION

Amorphous Compound A is so hygroscopic and so thermally unstable thatvarious troubles in producing the remedy are expected, and is hencedifficult to treat as material for the pharmaceutical preparation.Accordingly, there is a need to provide a thermally stable crystalline(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid salt having a long storage-life.

If crystalline Compound A is prepared according to the conventionalrecrystallization or salt-making process in which Compound A isconverted from a free form into the desired form, the product oftenprecipitates in the form of too fine crystallites to filter oroccasionally in the form of agar-like jelly. Further, since Compound Ais very hygroscopic, moisture in air causes to conversion of Compound Afrom the crystal state into the amorphous state and, as a result, thefilterability of the product lowers.

In the industrial preparation of Compound A, purification by columnchromatography is a troublesome procedure. Accordingly, it is alsodesired to provide a simple purification process for Compound A.

When Compound A is prepared in an industrial scale, it is important topurify Compound A in the free acid form. As described hereinafter, it isvery simple and industrially advantageous to prepare Compound A in thefree acid form from the after-described organic amine salt. In addition,it makes the preparation process further simple and advantageous thatCompound A in an ester form is hydrolyzed with a basic sodium orpotassium compound to convert directly into a sodium or potassium salt.

The present invention has an object to provide a thermally stablecrystalline sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylatehaving a long storage-life. The invention also has objects to provide asimple and industrially advantageous process for preparing crystallitesof the above-mentioned compound and to provide a salt of the compoundwith an organic amine employable in the process.

The invention relates to crystalline sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylatehaving the following characteristics:

DSC: an exothermic peak observed at a temperature in the range of 170 to175° C. with weight decrease; and

characteristic absorption bands of infrared absorption spectrum (KBrtablet): 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365,1310, 1260, 1110, 890 cm^(×1).

The invention also relates to crystalline potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylatehaving the following characteristics:

DSC: an exothermic peak observed at 177° C. with weight decrease; and

characteristic absorption bands of infrared absorption spectrum (KBrtablet): 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300,1240, 1110, 895 cm⁻¹.

The invention further relates to a process for preparation ofcrystalline sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate,comprising the following steps (1) to (6):

(1) hydrolyzing(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid in an ester form, to obtain(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid,

(2) causing the carboxylic acid obtained in the preceding step reactwith an organic amine, to prepare a salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid with the organic amine,

(3) adding an acid to the salt obtained in the preceding step, to obtain(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid,

(4) causing the carboxylic acid obtained in the preceding step reactwith a basic sodium or potassium compound in a mixed solvent of waterand an aliphatic alcohol or acetone, to obtain a sodium or potassiumsalt,

(5) recrystallizing the sodium or potassium salt obtained in thepreceding step using an aliphatic alcohol, and

(6) drying the product recrystallized in the preceding step underreduced pressure.

The invention furthermore relates to a process for preparation ofcrystalline sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate,comprising the following steps (1) to (4):

(1) causing(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid in an ester form react with a basic sodium or potassium compound,to obtain sodium or potassium(2S,3S)-3-[[1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate,

(2) crystallizing the sodium or potassium salt obtained in the precedingstep from a mixed solvent of water and acetone, to obtain crystallinesodium or potassium salt,

(3) recrystallizing the sodium or potassium salt obtained in thepreceding step using an aliphatic alcohol, and

(4) drying the product recrystallized in the preceding step underreduced pressure.

The invention still further relates to a salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid with an organic amine.

The invention further relates to a salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylicacid with an organic amine represented by the formula of:

(R¹)(R²)(R³)N

in which R¹ is hydrogen or a linear-chain, branched-chain or cyclicalkyl group having 1 to 10 carbon atoms; R² is hydrogen or alinear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10carbon atoms; and R³ is a linear-chain, branched-chain or cyclic alkylgroup of 1 to 10 carbon atoms which may have a substituent groupselected from the group consisting of halogen atoms, nitro, hydroxyl,carboxyl, guanidino, amino and aralkyl-amino groups; or otherwise R² andR³ can be combined to form a 5- to 7-membered ring comprising thenitrogen atom to which R² and R³ are connected (the ring may containadditional nitrogen atom).

The invention further relates to a salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylicacid with an organic amine selected from the group consisting ofpiperazine, adamantane amines, cyclohexylamine, dicyclohexylamine,tris(hydroxymethyl) aminomethane, arginine, lysine, benzathine andmeglumine.

MOST PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is explained below in more detail.

Crystalline sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylateof the invention can be obtained according to the following flow chart.

(Flow Chart)

Process 1:

-   -   ester form (A)→free form (B)→salt with an organic amine (C)→free        form (D)→Na or K salt (E)→recrystallized product (F)→immediately        dried crystallites (G);    -   or otherwise

Process 2:

-   -   ester form (A)→Na or K salt (E)→recrystallized product        (F)→immediately dried crystallites (G).

Each step is explained below.

(Process 1) (1) Step of Ester Form (A)→Free Form (B)

In a mixed solvent of water and an aliphatic alcohol such as methanol,ethanol or isopropyl alcohol,(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylicester (A) is hydrolyzed in the presence of a base such as sodiumhydroxide or potassium hydroxide, and is then neutralized to obtain(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid [free form (B)].

In the above procedure, the starting(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicester is an ester with an aliphatic alcohol, preferably an ester with aprimary, secondary or tertiary alcohol having 1 to 6 carbon atoms, morepreferably an ester with ethanol, methanol, n-propyl alcohol orisopropyl alcohol.

For synthesizing the ester of the starting material,(2S,3S)-3-alkoxycabonyloxirane-2-carboxylic acid and(1S)-1-isobutoxymethyl-3-methylbutylamine are, for example, condensedaccording to the normal condensation process (e.g., DCC-HOSu method,acid chloride method, and acid anhydride method).

(2) Step of Free Form (B)→Salt with an Organic Amine (C)

The free form (B) prepared in Step (1) is caused to react with anorganic amine in a solvent. Examples of the solvents include aliphaticalcohols such as methanol, ethanol, n-propyl alcohol and isopropylalcohol; aliphatic ethers such as diethyl ether and diisopropyl ether;aliphatic esters such as ethyl acetate; aliphatic ketones such asacetone and ethyl methyl ketone; and water. They may be used singly ormay be mixed. In consideration of flammability and toxicity, the solventpreferably is ethyl acetate, acetone, water or a mixture thereof. Thus,the salt with a desired organic amine can be obtained.

The organic amine is, for example, represented by the formula of:

(R¹)(R²)(R³)N

in which R¹ is hydrogen or a linear-chain, branched-chain or cyclicalkyl group having 1 to 10 carbon atoms; R² is hydrogen or alinear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10carbon atoms; and R³ is a linear-chain, branched-chain or cyclic alkylgroup (including a crosslinked cyclic hydrocarbon group such asadamantyl) having 1 to 10 carbon atoms which may have a substituentselected from the group consisting of halogen atoms, nitro, hydroxyl,carboxyl, guanidino, amino and aralkylamino groups; or otherwise R² andR³ can be combined to form a 5- to 7-membered ring comprising thenitrogen atom to which R² and R³ are connected (the ring may containadditional nitrogen atom). In consideration of cost, toxicity andcrystallization, the organic amine preferably is piperazine, anadamantane amine, cyclohexylamine, dicyclohexylamine,tris(hydroxymethyl)aminomethane, arginine (preferably in L-form), lysine(preferably in D-form or DL-form), benzathine or meglumine. The saltswith D- or DL-lysine, benzathine, meglumine andtris(hydroxymethyl)aminomethane can be immediately used as medicamentswithout further processing.(3) Step of Salt with an Organic Amine (C)→Free Form (D)

The salt with an organic amine (C) obtained in Step (2) is treated withan acid (e.g., hydrochloric acid) in the conventional manner, to obtain(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (D) of high purity. Thus obtained free form (D) gave no peakattributed to cleavage products (9.8 minutes, see Example 14) while thepeak is given by the free form or the metal salt thereof obtained byhydrolyzing the ester form (A) purified by silica-gel columnchromatography.

(In the Case of Preparing a Sodium Salt) (4) Step of Free Form (D)→NaSalt (E)

The free form (D) is converted into a sodium salt using a sodium sourcein an organic solvent capable of dissolving the free form (D) or in awater-containing organic solvent. Examples of the sodium sources includesodium metal, sodium alkoxides such as sodium methoxide and sodiumethoxide, sodium hydroxide, sodium carbonate, and sodium organiccarboxylate.

The solvent preferably is a mixed solvent of water and methanol, ethanolor acetone, in consideration of flammability and toxicity. If the watercontent is in the range of 1 to 5%, the product is obtained in a highyield.

Thus obtained solid product is well filtered so that the product iscollected by conventional filtration.

(5) Step of Na salt (E)→recrystallized product (F)

The solid sodium salt (E) obtained in Step (4) is dissolved in analiphatic alcohol such as methanol at a temperature of form roomtemperature to 60° C., and then an organic solvent is added toprecipitate a crystalline product. The organic solvent preferably is amixed solvent of ethyl acetate, methanol and ethanol, in considerationof flammability and toxicity. If the mixing ratio of ethylacetate/alcohol is in the range of 10/1 to 5/1, the product is obtainedin a high yield.

Thus recrystallized product (F) is well filtered so that the product iscollected by conventional filtration.

(6) Recrystallized Product (F)→Immediately Dried Crystallites (G)

The recrystallized product (F) is collected by filtration in a stream ofnitrogen gas, and immediately dried to obtain, in a high yield,crystalline sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(G) of high purity having the following characteristics:

DSC (the same as that in Example 15): exothermic peak observed in therange of 170 to 175° C. with weight decrease; and

characteristic absorption bands of infrared absorption spectrum (KBrtablet): 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365,1310, 1260, 1110, 890 cm⁻¹.

The product preferably is in the form of white crystalline needles.

The crystalline Na salt of the invention (described in Example 9) wassubjected to a comparative stability test. In the test, the Na salt wasleft at 80° C. for three days together with both the substance obtainedby condensing the solution to dryness and the product collected byfiltration and left at room temperature after recrystallization. As aresult, it was found that the Na salt of the invention was superior tothe substances for comparison in storage stability (see Example 15).

(In the Case of Preparing a Potassium Salt) (7) Step of Free Form (D)→KSalt (E)

The free form (D) is converted into a potassium salt using a potassiumsource in an organic solvent capable of dissolving the free form (D) orin a water-containing organic solvent. Examples of the potassium sourcesinclude potassium metal, potassium alkoxides such as potassium methoxideand potassium ethoxide, potassium hydroxide, potassium carbonate, andpotassium organic carboxylate.

The solvent preferably is a mixed solvent of water and methanol, ethanolor acetone, in consideration of flammability and toxicity. If the watercontent is in the range of 1 to 5%, the product is obtained in a highyield.

Thus obtained solid product is well filtered so that the product iscollected by conventional filtration.

(8) Step of K Salt (E)→Recrystallized Product (F)

The solid potassium salt (E) obtained in Step (7) is dissolved in analiphatic alcohol such as methanol at a temperature from roomtemperature to 60° C., and then an organic solvent is added toprecipitate a crystalline product. The organic solvent preferably is amixed solvent of ethyl acetate, methanol and ethanol, in considerationof flammability and toxicity. If the mixing ratio of ethylacetate/alcohol is in the range of 30/1 to 10/1, the product is obtainedin a high yield.

Thus recrystallized product (F) is well filtered so that the product iscollected by conventional filtration.

(9) Recrystallized Product (F)→Immediately Dried Crystallites (G)

The recrystallized product (F) is collected by filtration in a stream ofnitrogen gas, and immediately dried to obtain, in a high yield,crystalline potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(G) of high purity having the following characteristics:

DSC (the same as that in Example 15): exothermic peak observed at 177°C. with weight decrease; and

characteristic absorption bands of infrared absorption spectrum (KBrtablet): 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300,1240, 1110, 895 cm¹.

The product is preferably in the form of white crystalline needles.

The crystalline K salt of the invention (described in Example 11) wassubjected to a stability test in which the K salt was left at 80° C. forthree days. As a result, it was found that the K salt of the inventionwas excellent in storage stability (see Example 15).

(In the Case of Preparing a Calcium or Lithium Salt)

Crystalline calcium or lithium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylateof high purity can be prepared in a high yield through the steps similarto the above-mentioned Steps (4) to (9).

(Process 2) (1) Ester Form (A)→Na or K Salt (E) [Preparation of Na Salt(E)]

The Na salt (E) can be obtained by causing the ester form (A) describedin Step (1) of Process 1 react with a basic sodium compound (e.g.,sodium carbonate, sodium hydroxide) to hydrolyze, by condensing thereaction mixture, and then by adding a water-compatible organic solventsuch as acetone to crystallize the salt. If the water content is in therange of 1 to 5%, the product is obtained in a high yield.

[Preparation of K Salt (E)]

The K salt (E) can be obtained by causing the ester form (A) react witha basic potassium compound (e.g., potassium carbonate, potassiumhydroxide) to hydrolyze, by condensing the reaction mixture, and then byadding a water-compatible organic solvent such as acetone to crystallizethe salt. If the water content is in the range of 1 to 5%, the productis obtained in a high yield.

(2) Na or K Salt (E)→Recrystallized Product (F)→Immediately DriedCrystallites (G)

The steps of Na salt (E)→recrystallized product (F)→immediately driedcrystallites (G) can be the same as Steps (5) and (6) of Process 1described above.

The steps of K salt (E)→recrystallized product (F) →immediately driedcrystallites (G) can be the same as Steps (8) and (9) of Process 1described above.

As described above, the purification process in which(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid is converted to a salt with an organic amine, as well as theprocess in which the ester form of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid is hydrolyzed with a basic sodium or potassium compound to preparedirectly a sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate,does not need to involve the step for purifying the ester form bysilica-gel column chromatography. They are, therefore, simple andindustrially advantageous processes for purifying(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid.

The crystalline sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylateof the invention has a long storage life and excellent thermalstability, and accordingly is useful as a material for pharmaceuticalpreparation.

Form thus obtained crystalline sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate,medicaments can be prepared.

For preparing the medicaments, the salt can be processed according tothe conventional manner to give, for example, tablets, granules, powder,capsules, suspension, injection or suppository. In the pharmaceuticalpreparation, normal additives such as excipient, disintegrator, binder,lubricant, dye and diluent are used.

EXAMPLES

The invention is further described by the following examples. They by nomeans restrict the invention.

Example 1 Preparation of Free Form (B)

Crude ethyl(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(164.54 g) synthesized according to the process described in Patentreference 1 was dissolved in ethanol (520 mL). While the solution wascooled with ice (at a temperature of 10° C. or below), 1 mol/L aqueoussodium hydroxide solution (522 mL) was dropped. After the solution wasstirred for 1.5 hours while the temperature was kept, ethanol wasremoved under reduced pressure. Water (340 mL) was added, and then themixture was filtered through Celite to remove insoluble materials. Themixture was washed twice with ethyl acetate (300 mL). While the aqueousportion was cooled with ice, 6 mol/L aqueous hydrochloric acid (110 mL)was dropped to adjust the pH value to 1 and then the mixture wasextracted twice with ethyl acetate (300 mL). The ethyl acetate portionwas washed with water (300 mL) and with aqueous saturated sodiumchloride solution (200 mL), and then dried over anhydrous sodiumsulfate. The solvent was distilled off under reduced pressure to obtain(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid (139.61 g, 93.1%) as orange oil. The NMR data were the same asthose in Example 6.

Example 2 Preparation 1 of Salt with Organic Amine (C)

(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid (139.61 g) was dissolved in ethyl acetate (630 mL). The solutionwas cooled with ice (at a temperature of 10° C. or below) andcyclohexylamine (48.19 g) dissolved in ethyl acetate (190 mL) was added.The resultant mixture was stirred overnight at room temperature. Theprecipitated crystalline product was collected by filtration, washedwith ethyl acetate, and dried in air to obtain white powderycyclohexylamine salt of(2S,3)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (165.22 g, 88.0%). The obtained product (1.0 g) was recrystallizedfrom ethanol/water-containing ethyl acetate, to prepare crystallineflakes (925 mg, 92.5%).

Melting point: 128° C.-129° C.;

IR (KBr) cm⁻¹: 3310, 2950, 2850, 1660, 1620, 1570, 1540, 1460, 1445,1380, 1360, 1340, 1295, 1250, 1220, 1120, 945, 890; and

NMR (CDCl₃) δ: 0.9-1.0 (12H, m), 1.2-1.8 (9H, m), 1.8-1.9 (3H, m),2.0-2.1 (2H, m), 2.9-3.1 (1H, m), 3.2-3.3 (2H, m), 3.26 (1H, d, J=2 Hz),3.39 (2H, d, J=4 Hz), 3.49 (1H, d, J=2 Hz), 4.1-4.2 (1H, m), 6.46 (1H,d, J=9 Hz), 7.7-8.5 (1H, broad s).

Example 3 Preparation 2 of Salt with Organic Amine (C)

(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid (6.59 g) was dissolved in diethyl ether (67 mL), and benzathine(2.75 g) dissolved in diethyl ether (18 mL) was added while the solutionwas stirred at room temperature. Ethanol (6 mL) was then added todissolve the separated oily product, and diethyl ether (84 mL) wasfurther added. While the solution was cooled with ice, it was stirredovernight. The precipitated crystalline product was collected byfiltration, washed with diethyl ether, and dried at room temperatureunder reduced pressure to obtain white powdery benzathine salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid (7.43 g, 79.6%). Theobtained product (2 g) was recrystallized from ethanol/water, to preparethe benzathine salt (1.8 g, 90.0%) in crystalline flakes.

Melting point: 63° C.;

IR (KBr) cm⁻¹: 3400, 3260, 2950, 2850, 1650, 1450, 1380, 1295, 1240,1110, 890, 740; and

NMR(CDCl₃) δ: 0.9-1.0 (12H, m), 1.3-1.5 (2H, m), 1.5-1.6 (1H, m),1.8-1.9 (1H, m), 3.1-3.2 (4H, m), 3.9-4.0 (2H, m), 4.1-4.2 (1H, m), 6.36(1H, d, J=9 Hz), 7.39 (5H, s).

Example 4 Preparation 3 of Salt with Organic Amine (C)

(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid (1.31 g) was dissolved in acetone (6 mL), and meglumine (0.85 g)dissolved in water (1.5 mL) was added while the solution was stirred atroom temperature. Acetone (14 mL) was further added, and then thesolution was stirred overnight at room temperature. The precipitatedcrystalline product was collected by filtration, washed with acetone,dried in air and further dried under reduced pressure at roomtemperature to obtain meglumine salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (1.28 g, 61.0%) in white crystalline plates.

Melting point: 96° C.-98° C.;

IR (KBr) cm⁻¹: 3300, 2950, 1660, 1620, 1590, 1460, 1390, 1305, 1250,1100, 1080, 1030, 890; and

NMR (D₂O) δ: 0.7-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m), 1.7-1.8(1H, m), 2.69 (3H, s), 3.1-3.6 (15H, m), 3.7-3.8 (3H, m), 4.0-4.1 (2H,m).

Example 5 Preparation 4 of Salt with Organic Amine (C)

(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid (660 mg) was dissolved in ethanol (3 mL), and DL-lysine (336 mg)dissolved in water (1.0 mL) was added while the solution was stirred atroom temperature. The solution was condensed under reduced pressure, andethanol (10 mL) was added. The obtained solution was stirred overnightat room temperature. The precipitated crystalline product was collectedby filtration, washed with ethanol, dried in air and further dried underreduced pressure at room temperature to obtain white powdery DL-lysinesalt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (783 mg, 78.6%).

IR (KBr) cm⁻¹: 2950, 1640, 1460, 1380, 1100, 890; and

NMR (D₂O) δ: 0.7-0.9 (12H, m), 1.2-1.9 (10H, m), 2.93 (2H, t, J=7 Hz),3.2-3.5 (6H, m), 3.66 (1H, t, J=6 Hz), 4.0-4.1 (1H, m).

Example 6 Preparation 1 of Free Form (D)

The cyclohexylamine salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (165.22 g) was added to a mixture of water (400 mL) and ethylacetate (400 mL), to prepare a suspension. While the suspension wascooled with ice (at a temperature of 10° C. or below), 3 mol/L aqueoushydrochloric acid (140 mL) was dropped to adjust the pH value to approx.3. The ethyl acetate portion was collected, and independently theaqueous portion was extracted with ethyl acetate (200 mL). The extractedliquid portion and the above-collected ethyl acetate portion were mixed,washed with water (200 mL) and with aqueous saturated sodium chloridesolution (200 mL), and dried over anhydrous sodium sulfate. The solventwas distilled off under reduced pressure to obtain(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (122.83 g, 99.9%) as yellow oil.

NMR (CDCl₃) δ: 0.9-1.0 (12H, m), 1.3-1.6 (3H, m), 1.8-1.9 (1H, m),3.1-3.3 (2H, m), 3.43 (2H, d, J=4 Hz), 3.47 (1H, d, J=2 Hz), 3.71 (1H,d, J=2 Hz), 4.1-4.2 (1H, m), 6.50 (1H, d, J=9 Hz), 9.60 (1H, broad s).

Example 7 Preparation 2 of Free Form (D)

In the same manner as Example 6,(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid was prepared as yellow oil from the amine salt such as thebenzathine salt, the meglumine salt or the DL-lysine salt. The NMR datawere the same as those in Example 6.

Example 8 Preparation of Na Salt (E)

(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid (48.59 g) was dissolved in acetone (245 mL), and sodium carbonate(8.51 g) dissolved in water (32.9 mL) was slowly added. The solution wasstirred at room temperature to 50° C. for three hours until thedeposited inorganic salt was dissolved. While the temperature was kept,acetone (200 mL) was dropped and a seed crystal was placed in thesolution, and then the solution was stirred for 30 minutes. Acetone (510mL) was again dropped, and the solution was stirred for 1 hour while thetemperature was kept. While the solution was gradually cooled to roomtemperature, it was stirred overnight. The precipitated crystallineproduct was collected by filtration, washed with a mixture of 3%water-acetone (70 mL), dried in air to obtain white solid sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(46.1 g, 87.9%). The NMR data of the solid product were the same asthose in Example 9.

Example 9 Preparations of Recrystallized Product (F) and ImmediatelyDried Na Crystallites (G)

The sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(25.18 g) prepared in Example 8 was dissolved in methanol (85 mL) at 50°C., and ethyl acetate (100 mL) was dropped while the temperature waskept. While the temperature was still kept, a seed crystal was placed inthe solution, the solution was stirred for 1 hour, and ethyl acetate(150 mL) was again dropped. While the temperature was still kept, ethylacetate (100 mL) was further dropped and furthermore ethyl acetate (75mL) was dropped, and then the solution was stirred for 1 hour. Whilegradually cooled to room temperature, the solution was stirredover-night. The precipitated crystalline product was collected byfiltration, washed with three portions of a mixture of ethylacetate/methanol (5/1, 40 mL, 25 mL, 25 mL) in a stream of nitrogen gas.Before completely dried in air, the crystalline product was dried underreduced pressure at 40° C. overnight to obtain sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(21.1 g, 83.9%) in white crystalline needles.

Melting point: 170° C.-175° C. (dec.);

IR (KBr) cm⁻¹: 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395,1365, 1310, 1260, 1110, 890; and

NMR (D₂O) δ: 0.7-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m), 1.7-1.8(1H, m), 3.1-3.3 (2H, m), 3.3-3.5 (4H, m), 4.0-4.1 (1H, m).

Example 10 Preparation of K Salt (E)

The(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid (4.83 g) prepared in Example 6 or 7 was dissolved in acetone (23mL), and potassium carbonate (1.11 g) dissolved in water (4.5 mL) wasslowly added. The solution was stirred at room temperature until thedeposited inorganic salt was dissolved. While the solution was heated at50° C. in a bath, acetone (100 mL) was dropped and a seed crystal wasplaced in the solution, and then the solution was stirred for 30minutes, Acetone (94 mL) was again dropped, and the solution was stirredfor 1 hour while the temperature was kept. While gradually cooled toroom temperature, the solution was stirred overnight. The precipitatedcrystalline product was collected by filtration, washed with a mixtureof 2% water-acetone (20 mL), dried in air to obtain white solidpotassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(4.28 g, 81.7%). The NMR data of the solid product were the same asthose in Example 11.

Example 11 Preparations of Recrystallized Product (F) and ImmediatelyDried K Crystallites (G)

The potassium(2S,3)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(500 mg) prepared in Example 10 was dissolved in methanol (3.5 mL) toprepare a solution. While the solution was heated at 50° C. in a bath,ethyl acetate (12 mL) was added and a seed crystal was placed in thesolution. While the temperature was kept, the solution was stirred for 1hour and ethyl acetate (23 mL) was again dropped. The solution wasstirred for 1 hour while the temperature was still kept. While graduallycooled to room temperature, the solution was stirred overnight. Theprecipitated crystallites were collected by filtration, washed with amixture of ethyl acetate/methanol (10/1, 10 mL) in a stream of nitrogengas. Before completely dried in air, the crystalline product was driedunder reduced pressure at 40° C. overnight to obtain potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(445 mg, 89.0%) in white crystalline needles.

Melting point: 177° C. (dec.);

IR (KBr) cm⁻¹: 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380,1300, 1240, 1110, 895; and

NMR (D₂O) δ: 0.8-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m), 1.7-1.8(1H, m), 3.2-3.3 (2H, m), (4H, m), 4.0-4.1 (1H, m).

Example 12 Preparation of Na Salt (E) (Process 2)

Crude ethyl(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(15.29 g) synthesized according to the process (acid chloride method)similar to that described in Patent reference 1 was dissolved in ethanol(24 mL). While the solution was stirred at room temperature, sodiumcarbonate (2.44 g) dissolved in water (24 mL) was dropped. After thesolution was stirred at room temperature for 1 hour, it was furtherstirred at 90° C. to 95° C. for 2 hours. The solvent was distilled offunder reduced pressure, and water (50 mL) was added to the residue. Theobtained solution was washed with two portions of ethyl acetate (50 mL),and water was distilled off under reduced pressure. Acetone (400 mL) wasdropped to the residue, and the obtained solution was stirred overnightat room temperature. The precipitated crystalline product was collectedby filtration, washed with a mixture of 3% water-acetone (30 mL), washedagain with acetone (30 mL), and dried in air to obtain white solidsodium(2S,3S)-3[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(10.55 g, 70.3%). The NMR data of the solid product were the same asthose in Example 9.

Example 13 Preparation of Na Salt (E) (Process 2)

Crude ethyl(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(7.82 g) synthesized according to the process (acid chloride method)similar to that described in Patent reference 1 was dissolved in ethanol(24 mL). While the solution was cooled with ice and stirred, 1 mol/Laqueous solution of sodium hydroxide (23.55 mL) was dropped. After thesolution was stirred for 1.5 hours while the temperature was kept, thesolvent was distilled off under reduced pressure. Water (39 mL) wasadded to the residue, and the obtained solution was washed with twoportions of ethyl acetate (39 mL), and then water was distilled offunder reduced pressure. Acetone (157 mL) was added to the residue, andthe solution was stirred overnight at room temperature. The precipitatedcrystalline product was collected by filtration, washed with a mixtureof 3% water-acetone (30 mL), washed again with acetone (30 mL), anddried in air to obtain white solid sodium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(5.36 g, 69.9%). The NMR data of the solid product were the same asthose in Example 9.

Example 14 Purity Test on Free Form (D)

The products in the free form (D) obtained in Examples 6 and 7 weresubjected to a purity test. In the test, each free form (D) gave no peakattributable to cleavage products (9.8 minutes) under thebelow-described HPLC conditions, while the peak was given by the freeform or the metal salt thereof obtained by hydrolyzing the ester form(A) purified by a silica-gel column chromatography.

(HPLC Conditions)

column: YMC-A302 (150×4.5 mm),

mobile phase: a mixture of 0.1 mol/L sodium dihydrogen phosphate reagent(pH: 3.0)/acetonitrile (5:2),

flow rate: controlled so that the retention time of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylicacid would be approx. 13 minutes, and

detector: UV (210 nm).

Example 15 Comparative Stability Test (Procedure)

As a sample, 10 mg of sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylatewas weighed and placed in a test tube. After the sample was kept in athermostat at 80° C. for three days under a light-shaded and airtightcondition, the amount of remaining substance was measured by HPLC.

(HPLC Conditions)

The same as those in Example 14.

(Conditions of DSC: Differential Scanning Calorimetry)

Each of the sodium or potassium(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate(sample) and 0.01 g of α-alumina (standard) was placed in a container,and heated at temperatures elevating from room temperature to approx.200° C. at the heating rate of 2° C./minute. (A differential scanningcalorimetric thermobalance [TAS100, TG-DCS type, Rigaku Corporation] wasused.)

(Results)

The results are set forth in Table 1.

TABLE 1 Remaining ratio (%) after 3 DSC days at 80° C. Ref. Na salt   157° C. 13-77 amor. > crys³⁾ (obtained according to the conventionalprocess¹⁾) Na salt 160-170° C. 38-77 amor. > crys³⁾ (obtained throughrecrystallization and drying in air²⁾) Na salt 170-175° C. 95-98crystalline needles (obtained in Example 9) K salt    177° C. 99.9crystalline needles (obtained in Example 11) ¹⁾obtained by condensationto dryness; ²⁾recrystallized and collected by filtration, and then leftat room temperature; and ³⁾presumed to be more in amorphous state thanin crystal state.

The results in Table 1 clearly indicate that the crystalline Na saltprepared in Example 9 (in which the salt was recrystallized, collectedby filtration in a stream of nitrogen gas and immediately dried) wassuperior in storage stability to the salt obtained by condensation todryness (according to the conventional process) or obtained throughrecrystallization and drying in air. Further, the results in Table 1indicate that the crystalline K salt prepared in Example 11 also hadexcellent storage stability.

1. A salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid with an organic amine.
 2. A salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid with an organic amine represented by the formula of:(R¹)(R²)(R³)N in which R¹ is hydrogen or a linear-chain, branched-chainor cyclic alkyl group having 1 to 10 carbon atoms; R² is hydrogen or alinear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10carbon atoms; and R³ is a linear-chain, branched-chain or cyclic alkylgroup having 1 to 10 carbon atoms which may have a substituent selectedfrom the group consisting of halogen atoms, nitro, hydroxyl, carboxyl,guanidino, amino and aralkylamino groups; or otherwise R² and R³ can becombined to form a 5- to 7-membered ring comprising the nitrogen atom towhich R² and R³ are connected, the ring possibly containing additionalnitrogen atom.
 3. A salt of(2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylicacid with an organic amine selected from the group consisting ofpiperazine, adamantane amines, cyclohexylamine, dicyclohexylamine,tris(hydroxymethyl)aminomethane, arginine, lysine, benzathine, andmeglumine.